Modulation of immunostimulatory activity of immunostimulatory oligonucleotide analogs by positional chemical changes

ABSTRACT

The invention relates to the therapeutic use of oligonucleotides or oligonucleotide analogs as immunostimulatory agents in immunotherapy applications. The invention provides methods for enhancing the immune response caused by immunostimulatory oligonucleotide compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/712,898, filed on Nov. 15, 2000. Thisapplication also claims priority from U.S. provisional patentapplication Ser. Nos. 60/235,452 and 60/235,453, both filed on Sep. 26,2000. Each of the patent applications listed above is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates to the therapeutic use of oligonucleotidesor oligonucleotide analogs as immunostimulatory agents in immunotherapyapplications.

[0004] 2. Summary of the Related Art

[0005] Oligonucleotides have become indispensable tools in modernmolecular biology, being used in a wide variety of techniques, rangingfrom diagnostic probing methods to PCR to antisense inhibition of geneexpression and immunotherapy applications. This widespread use ofoligonucleotides has led to an increasing demand for rapid, inexpensiveand efficient methods for synthesizing oligonucleotides. The synthesisof oligonucleotides for antisense and diagnostic applications can now beroutinely accomplished. See e.g., Methods in Molecular Biology, Vol 20:Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, Ed.,Humana Press, 1993); Oligonucleotides and Analogues: A PracticalApproach, pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman,supra. Agrawal and Iyer, Curr. Op. in Biotech. 6: 12 (1995); andAntisense Research and Applications (Crooke and Lebleu, Eds., CRC Press,Boca Raton, 1993). Early synthetic approaches included phosphodiesterand phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72: 209(1972) discloses phosphodiester chemistry for oligonucleotide synthesis.Reese, Tetrahedron Lett. 34: 3143-3179 (1978), discloses phosphotriesterchemistry for synthesis of oligonucleotides and polynucleotides. Theseearly approaches have largely given way to the more efficientphosphoramidite and H-phosphonate approaches to synthesis. Beaucage andCaruthers, Tetrahedron Lett. 22: 1859-1862 (1981), discloses the use ofdeoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawaland Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimizedsynthesis of oligonucleotides by the H-phosphonate approach.

[0006] Both of these modern approaches have been used to synthesizeoligonucleotides having a variety of modified internucleotide linkages.Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teachessynthesis of oligonucleotide methylphosphonates using phosphoramiditechemistry. Connolly et al., Biochemistry 23: 3443 (1984), disclosessynthesis of oligonucleotide phosphorothioates using phosphoramiditechemistry. Jager et al., Biochemistry 27: 7237 (1988), disclosessynthesis of oligonucleotide phosphoramidates using phosphoramiditechemistry. Agrawal et al., Proc. Natl. Acad. Sci. USA 85: 7079-7083(1988), discloses synthesis of oligonucleotide phosphoramidates andphosphorothioates using H-phosphonate chemistry.

[0007] More recently, several researchers have demonstrated the validityof the use of oligonucleotides as immunostimulatory agents inimmunotherapy applications. The observation that phosphodiester andphosphorothioate oligonucleotides can induce immune stimulation hascreated interest in developing this side effect as a therapeutic tool.These efforts have focused on phosphorothioate oligonucleotidescontaining the dinucleotide CpG.

[0008] Kuramoto et al., Jpn. J. Cancer Res. 83: 1128-1131 (1992) teachesthat phosphodiester oligonucleotides containing a palindrome thatincludes a CpG dinucleotide can induce interferon-alpha and gammasynthesis and enhance natural killer activity. Krieg et al., Nature 371:546-549 (1995) discloses that phosphorothioate CpG-containingoligonucleotides are immunostimulatory. Liang et al., J. Clin. Invest.98: 1119-1129 (1996) discloses that such oligonucleotides activate humanB cells.

[0009] Pisetsky, D. S.; Rich C. F., Life Sci. 54: 101 (1994), teachesthat the immunostimulatory activity of CpG-oligos is further enhanced bythe presence of phosphorothioate (PS) backbone on these oligos.Tokunaga, T.; Yamamoto, T.; Yamamoto, S. Jap. J. Infect. Dis. 52:1(1999), teaches that immunostimulatory activity of CpG-oligos isdependent on the position of CpG-motif and the sequences flankingCpG-motif. The mechanism of activation of immune stimulation byCpG-oligos has not been well understood. Yamamoto, T.; Yamamoto, S.;Kataoka, T.; Tokunaga, T., Microbiol. Immunol. 38:831 (1994), however,suggests that CpG-oligos trigger immune cascade by binding to anintracellular receptor/protein, which is not characterized yet.

[0010] Several researchers have found that this ultimately triggersstress kinase pathways, activation of NF-κB and induction of variouscytokines such as IL-6, IL-12, γ-IFN, and TNF-α. (See e.g., Klinman, D.M.; Yi, A. K.; Beaucage, S. L.; Conover, J.; Krieg, A. M., Proc. Natl.Acad. Sci. U.S.A. 93: 2879 (1996); Sparwasser, T.; Miethke, T.; Lipford,G. B.; Erdmann, A.; Haecker, H.; Heeg, K.; Wagner, H., Eur. J. Immunol.27:1671 (1997); Lipford, G. B.; Sparwasser, T.; Bauer, M.; Zimmermann,S.; Koch, E. S.; Heeg, K.; Wagner, H. Eur. J., Immunol. 27: 3420 (1997);Sparwasser, T.; Koch, E. S.; Vabulas, R. M.; Lipford, G. B.; Heeg, K.;Ellart, J. W.; Wagner, H., Eur. J. Immunol. 28: 2045 (1998); and Zhao,Q.; Temsamani, J.; Zhou, R. Z.; Agrawal, S. Antisense Nucleic Acid DrugDev. 7: 495 (1997).)

[0011] The use of CpG-PS-oligos as antitumor, antiviral, antibacterialand antiinflammatory agents and as adjuvants in immunotherapy has beenreported. (See e.g., Dunford, P. J.; Mulqueen, M. J.; Agrawal, S.Antisense 97: Targeting the Molecular Basis of Disease, (NatureBiotechnology) Conference abstract, 1997, pp 40; Agrawal, S.; KandimallaE. R. Mol. Med. Today 6: 72 (2000); Chu. R. S.; Targoni, O. S.; Krieg,A. M.; Lehmann, P. V.; Harding, C. V. J. Exp. Med. 186:1623 (1997);Zimmermann, S.; Egeter, O.; Hausmann, S.; Lipford, G. B.; Rocken, M.;Wagner, H.; Heeg, K. J. Immunol. 160: 3627 (1998).) Moldoveanu et al.,Vaccine 16: 1216-124 (1998) teaches that CpG-containing phosphorothioateoligonucleotides enhance immune response against influenza virus.McCluskie and Davis, J. Immunol. 161: 4463-4466 (1998) teaches thatCpG-containing oligonucleotides act as potent adjuvants, enhancingimmune response against hepatitis B surface antigen.

[0012] Zhao, Q.; Temsamani, J.; Idarola, P.; Jiang, Z.; Agrawal, S.Biochem. Pharmacol. 51: 173 (1996), teaches that replacement ofdeoxynucleosides in a CpG-motif with 2′-O-methylribonucleosidessuppresses immunostimulatory activity, suggesting that a rigid C3′-endoconformation induced by 2′-O-methyl modification does not allow properrecognition and/or interaction of CpG-motif with the proteins involvedin the immunostimulatory pathway. This reference further teaches thatsubstitution of a methyl group for an unbridged oxygen on the phosphategroup between C and G of a CpG-motif suppresses immune stimulatoryactivity, suggesting that negative charge on phosphate group isessential for protein recognition and interaction.

[0013] Zhao, Q.; Yu, D.; Agrawal, S. Bioorg. Med. Chem. Lett. 9:3453(1999), teaches that substitution of one or two 2′-deoxynucleosidesadjacent to CpG-motif with 2′- or 3′-O-methylribonucleosides on the5′-side causes a decrease in immunostimulatory activity, while the samesubstitutions have insignificant effect when they were placed on the3′-side of the CpG-motif. However, Zhao, Q.; Yu, D.; Agrawal, S. Bioorg.Med. Chem. Lett. 10: 1051 (2000), teaches that the substitution of adeoxynucleoside two or three nucleosides away from the CpG-motif on the5′-side with one or two 2′-O-methoxyethyl- or 2′- or3′-O-methylribonucleosides results in a significant increase inimmunostimulatory activity.

[0014] The precise structural requirements and specific functionalgroups of CpG-motif necessary for the recognition of protein/receptorfactor that is responsible for immune stimulation have not yet beenstudied in detail. There is, therefore, a need for new immunostimulatorymotifs which may provide improved immunostimulatory activity.

BRIEF SUMMARY OF THE INVENTION

[0015] The invention provides methods for enhancing the immune responsecaused by immunostimulatory oligonucleotide compounds. The methodsaccording to the invention enable increasing the immunostimulatoryeffect for immunotherapy applications. Thus, the invention furtherprovides methods for making and using such oligonucleotide compounds.

[0016] The present inventors have surprisingly discovered thatpositional modification of immunostimulatory oligonucleotidesdramatically affects their immunostimulatory capabilities. Inparticular, modifications in the immunostimulatory domain and/or thepotentiation domain enhance the immunostimulatory effect in areproducible and predictable manner.

[0017] In a first aspect, the invention provides immunostimulatoryoligonucleotide compounds comprising an immunostimulatory domain and,optionally, one or more potentiation domains. In some embodiments, theimmunostimulatory domain comprises a dinucleotide analog that includes anon-naturally occurring pyrimidine base. In some embodiments, theimmunostimulatory domain and/or the potentiation domain include animmunostimulatory moiety at a specified position, as describedhereinbelow. In some embodiments, the immunostimulatory oligonucleotidecomprises a 3′-3′ linkage. In one embodiment, such 3′-3′ linkedoligonucleotides have two accessible 5′-ends.

[0018] In a second aspect, the invention provides methods for modulatingthe immunostimulatory effect of an immunostimulatory oligonucleotidecompound. In some embodiments, the method comprises introducing into theimmunostimulatory domain a dinucleotide analog that includes anon-naturally occurring pyrimidine base. In some embodiments, the methodcomprises introducing into the immunostimulatory domain and/orpotentiation domain an immunostimulatory moiety at a specified position,as described hereinbelow. In some embodiments, the method comprisesintroducing into the oligonucleotide a 3′-3′ linkage.

[0019] In a third aspect, the invention provides methods for generatingan immune response in a patient, such methods comprising administeringto the patient an immunostimulatory oligonucleotide compound accordingto the invention.

[0020] In a fourth aspect, the invention provides methods fortherapeutically treating a patient having disease caused by a pathogen,such methods comprising administering to the patient animmunostimulatory oligonucleotide compound according to the invention.

[0021] In a fifth aspect, the invention provides methods for treating acancer patient, such methods comprising administering to the patient animmunostimulatory oligonucleotide compound according to the invention.

[0022] In a sixth aspect, the invention provides methods for treatingautoimmune disorders, such as autoimmune asthma, such methods comprisingadministering to the patient an oligonucleotide analog immunostimulatorycompound according to the invention. Administration is carried out asdescribed for the third aspect of the invention.

[0023] In a seventh aspect, the invention provides methods for treatingairway inflammation or allergies, such methods comprising administeringto the patient an oligonucleotide analog immunostimulatory compoundaccording to the invention. Administration is carried out as describedfor the third aspect of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0024]FIG. 1 shows results of proliferation assays usingoligonucleotides having 1′,2′-dideoxyribose substitutions at variouspositions.

[0025]FIG. 2 shows results of spleen weight assays usingoligonucleotides having 1′,2′-dideoxyribose substitutions at variouspositions.

[0026]FIG. 3 shows results of proliferation assays using differentoligonucleotides having 1′,2′-dideoxyribose substitutions at variouspositions.

[0027]FIG. 4 shows results of spleen weight assays using differentoligonucleotides having 1′,2′-dideoxyribose substitutions at variouspositions.

[0028]FIG. 5 shows results of proliferation assays usingoligonucleotides having C3-linker substitutions at various positions.

[0029]FIG. 6 shows results of spleen weight assays usingoligonucleotides having C3-linker substitutions at various positions.

[0030]FIG. 7 shows results of proliferation assays usingoligonucleotides having Spacer 9 or Spacer 18 substitutions at variouspositions.

[0031]FIG. 8 shows results of spleen weight assays usingoligonucleotides having Spacer 9 or Spacer 18 substitutions at variouspositions.

[0032]FIG. 9 shows results of proliferation assays usingoligonucleotides having amino-linker substitutions at various positions.

[0033]FIG. 10 shows results of spleen weight assays usingoligonucleotides having amino-linker substitutions at various positions.

[0034]FIG. 11 shows results of proliferation assays usingoligonucleotides having 3′-deoxynucleoside substitutions at variouspositions.

[0035]FIG. 12 shows results of spleen weight assays usingoligonucleotides having 3′-deoxynucleoside substitutions at variouspositions.

[0036]FIG. 13 shows results of proliferation assays usingoligonucleotides having methylphosphonate substitutions at variouspositions.

[0037]FIG. 14 shows results of spleen weight assays usingoligonucleotides having methylphosphonate substitutions at variouspositions.

[0038]FIG. 15 shows results of proliferation assays usingoligonucleotides having 2′-O-methylribonucleoside or 2′-O-methoxyethylsubstitutions at various positions.

[0039]FIG. 16 shows results of spleen weight assays usingoligonucleotides having 2′-O-methylribonucleoside or 2′-O-methoxyethylsubstitutions at various positions.

[0040]FIG. 17 shows results of proliferation assays usingoligonucleotides having 5′-3′,5′-5′, or 3′-3′ linkage substitutions atvarious positions.

[0041]FIG. 18 shows results of spleen weight assays usingoligonucleotides having P-L-deoxynucleotide substitutions at variouspositions.

[0042]FIG. 19 shows results of spleen weight assays usingoligonucleotides having 2′-O-propargyl substitutions at variouspositions.

[0043]FIG. 20 shows results of spleen weight assays usingoligonucleotides having various substitutions at various positions.

[0044]FIG. 21 shows results of spleen weight assays usingoligonucleotides having 7-deazaguanine substitution within theimmunostimulatory dinucleotide.

[0045]FIG. 22 shows results of proliferation assays usingoligonucleotides having 6-thioguanine substitution within theimmunostimulatory dinucleotide.

[0046]FIG. 23 shows results of spleen weight assays usingoligonucleotides having 5-hydroxycytosine or N4-ethylcytosinesubstitution within the immunostimulatory dinucleotide.

[0047]FIG. 24 shows results of spleen weight assays usingoligonucleotides having 5-hydroxycytosine or N4-ethylcytosinesubstitution within the immunostimulatory dinucleotide.

[0048]FIG. 25 shows results of proliferation assays usingoligonucleotides having arabinofuranosylcytosine (aracytidine; Ara-C)substitution within the immunostimulatory dinucleotide.

[0049]FIG. 26 shows results of spleen weight assays usingoligonucleotides having 4-thiouracil substitution within theimmunostimulatory dinucleotide.

[0050]FIG. 27 shows the chemical structure of a CpG-motif, showingfunctional groups on cytosine that serve as hydrogen bond acceptor andhydrogen bond donor groups.

[0051]FIG. 28 shows the chemical structures of cytosine (1) and cytosineanalogs (2-7). In the nucleosides cytidine, deoxycytidine, and relatedanalogs, the substituent R is ribose or 2′-deoxyribose.

DETAILED DESCRIPTION

[0052] The invention relates to the therapeutic use of oligonucleotidesand oligonucleotide analogs as immunostimulatory agents forimmunotherapy applications. The patents and publications cited hereinreflect the level of knowledge in the field and are hereby incorporatedby reference in their entirety. In the event of conflict between anyteaching of any reference cited herein and the present specification,the latter shall prevail, for purposes of the invention.

[0053] The invention provides methods for enhancing the immune responsecaused by immunostimulatory oligonucleotide compounds for immunotherapyapplications. Thus, the invention further provides compounds havingoptimal levels of immunostimulatory effect for immunotherapy and methodsfor making and using such oligonucleotide compounds.

[0054] The present inventors have surprisingly discovered thatpositional chemical modifications introduced in immunostimulatoryoligonucleotides dramatically affect their immunostimulatorycapabilities. In particular, modifications in the immunostimulatorydomain and/or the potentiation domain can enhance the immunostimulatoryeffect in a reproducible manner for desired applications.

[0055] In a first aspect, the invention provides immunostimulatoryoligonucleotide compounds comprising an immunostimulatory domain and,optionally, one or more potentiation domains. In certain preferredembodiments, the immunostimulatory domain comprises a dinucleotideanalog that includes a non-natural pyrimidine nucleoside.

[0056] For purposes of all aspects of the invention, the term“oligonucleotide” includes polymers of two or more deoxyribonucleosides,or any modified nucleoside, including 2′- or 3′-substituted nucleosides,2′- or 3′-O-substituted ribonucleosides, deazanucleosides, or anycombination thereof. Such monomers may be coupled to each other by anyof the numerous known internucleoside linkages. In certain preferredembodiments, these internucleoside linkages may be phosphodiester,phosphotriester, phosphorothioate, phosphorodithioate, orphosphoramidate linkages, including 3′-5′,2′-5′,3′-3′, and 5′-5′linkages of any of the foregoing, or combinations thereof. The termoligonucleotide also encompasses such polymers having chemicallymodified bases or sugars and/or having additional substituents,including without limitation lipophilic groups, intercalating agents,diamines and adamantane. The term oligonucleotide also encompassespeptide nucleic acids (PNA), peptide nucleic acids with phosphate groups(PHONA), locked nucleic acids (LNA), morpholinonucleic acids, andoligonucleotides comprising non-pentose sugar (e.g. hexose) or abasicsugar backbones or backbone sections, as well as oligonucleotides thatinclude backbone sections with non-sugar linker or spacer groups, asfurther described hereinbelow.

[0057] For purposes of the invention the terms “2′-substituted” and“3′-substituted” mean (respectively) substitution of the 2′ (or 3′)position of the pentose moiety with a halogen (preferably Cl, Br, or F),or an —O-lower alkyl group containing 1-6 saturated or unsaturatedcarbon atoms, or with an —O-aryl or allyl group having 2-6 carbon atoms,wherein such alkyl, aryl or allyl group may be unsubstituted or may besubstituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro,acyl, acyloxy, alkoxy, carboxyl, carbalkoxy, or amino groups; or such 2′substitution may be with a hydroxy group (to produce a ribonucleoside)or an amino group, but not with a 2′ (or 3′) H group.

[0058] For purposes of the invention, the term “immunostimulatoryoligonucleotide compound” means a compound comprising animmunostimulatory dinucleotide, without which the compound would nothave an immunostimulatory effect. An “immunostimulatory dinucleotide” isa dinucleotide having the formula 5′-pyrimidine-purine-3′, wherein“pyrimidine” is a natural or non-natural pyrimidine nucleoside and“purine” is a natural or non-natural purine nucleoside. One suchimmunostimulatory dinucleotide is CpG. The terms “CpG” and “CpGdinucleotide” refer to the dinucleotide5′-deoxycytidine-deoxyguanosine-3′, wherein p is an internucleotidelinkage, preferably selected from the group consisting ofphosphodiester, phosphorothioate, and phosphorodithioate.

[0059] For purposes of the invention, a “dinucleotide analog” is animmunostimulatory dinucleotide as described above, wherein either orboth of the pyrimidine and purine nucleosides is a non-naturalnucleoside. A “non-natural” nucleoside is one that includes anon-naturally occurring base and/or a non-naturally occurring sugarmoiety. For purposes of the invention, a base is considered to benon-natural if it is not selected from the group consisting of thymine,guanine, cytosine, adenine, and uracil. The terms “C*pG” and “CpG*”refer to immunostimulatory dinucleotide analogs comprising a cytidineanalog (non-natural pyrimidine nucleoside) or a guanosine analog(non-natural purine nucleoside), respectively.

[0060]FIG. 27 shows the chemical structure of a CpG-motif, showing thefunctional groups on cytosine that serve as hydrogen bond acceptor andhydrogen bond donor groups. Cytosine has two hydrogen bond acceptorgroups at positions 2 (keto-oxygen) and 3 (nitrogen), and a hydrogenbond donor group at the 4-position (amino group) These groups can serveas potential recognizing and interacting groups with receptors that areresponsible for immune stimulation. FIG. 28 shows cytosine analogs thatare isostructural with natural cytosine, including5-methyl-deoxycytosine (2), 5-methyl-deoxyisocytosine (3),5-hydroxy-deoxycytosine (4), deoxyuridine (5), N4-ethyl-deoxycytosine(6), and deoxy-P-base (7).

[0061] In one embodiment, therefore, the immunostimulatory dinucleotidecomprises a pyrimidine nucleoside of structure (I):

[0062] wherein D is a hydrogen bond donor, D′ is selected from the groupconsisting of hydrogen, hydrogen bond donor, hydrogen bond acceptor,hydrophilic group, hydrophobic group, electron withdrawing group andelectron donating group, A is a hydrogen bond acceptor, X is carbon ornitrogen, and S is a pentose or hexose sugar ring linked to thepyrimidine base. In some embodiments, the pyrimidine nucleoside is anon-natural pyrimidine nucleoside, i.e., the compound of structure (I)is not cytidine or deoxycytidine.

[0063] In some embodiments, the base moiety in (I) is a non-naturallyoccurring pyrimidine base. Examples of preferred non-naturally occurringpyrimidine bases include, without limitation, 5-hydroxycytosine,5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4-ethylcytosine,and 4-thiouracil. In some embodiments, the sugar moiety S in (1) is anon-naturally occurring sugar moiety. For purposes of the presentinvention, a “naturally occurring sugar moiety” is ribose or2′-deoxyribose, and a “non-naturally occurring sugar moiety” is anysugar other than ribose or 2′-deoxyribose that can be used in thebackbone for an oligonucleotide. Arabinose and arabinose derivatives areexamples of a preferred non-naturally occurring sugar moieties.

[0064] Immunostimulatory domains according to the invention may includeimmunostimulatory moieties on one or both sides of the immunostimulatorynatural dinucleotide or non-natural dinucleotide analog. For example, animmunostimulatory domain could be depicted as 5′ - - -X1-X2-Y-Z-X3-X4 - - - 3′

[0065] wherein Y represents cytidine or a non-natural pyrimidinenucleoside analog, Z represents guanosine or a non-natural purinenucleoside analog, and each X independently represents a nucleoside oran immunostimulatory moiety according to the invention. An“immunostimulatory moiety” is a chemical structure at a particularposition within the immunostimulatory domain or the potentiation domainthat causes the immunostimulatory oligonucleotide to be moreimmunostimulatory than it would be in the absence of theimmunostimulatory moiety.

[0066] Preferred immunostimulatory moieties include modifications in thephosphate backbones including without limitation methylphosphonates,methylphosphonothioates phosphotriesters, phosphothiotriestersphosphorothioates, phosphorodithioates, triester prodrugs, sulfones,sulfonamides, sulfamates, formacetal, N-methylhydroxylamine, carbonate,carbamate, boranophosphonate, phosphoramidates, especially primaryamino-phosphoramidates, N3 phosphoramidates and N5 phosphoramidates, andstereospecific linkages (e.g., (R)- or (S)-phosphorothioate,alkylphosphonate, or phosphotriester linkages). Preferredimmunostimulatory moieties according to the invention further includenucleosides having sugar modifications, including without limitation2′-substituted pentose sugars including without limitation2′-O-methylribose, 2′-O-methoxyethylribose, 2′-O-propargylribose, and2′-deoxy-2′-fluororibose; 3′-substituted pentose sugars, includingwithout limitation 3′-O-methylribose; 1′,2′-dideoxyribose; hexosesugars, including without limitation arabinose, 1′-methylarabinose,3′-hydroxymethylarabinose, 4′-hydroxymethylarabinose, and 2′-substitutedarabinose sugars; and alpha-anomers.

[0067] Preferred immunostimulatory moieties according to the inventionfurther include oligonucleotides having other carbohydrate backbonemodifications and replacements, including peptide nucleic acids (PNA),peptide nucleic acids with phosphate groups (PHONA), locked nucleicacids (LNA), morpholinonucleic acids, and oligonucleotides havingbackbone sections with alkyl linkers or amino linkers. The alkyl linkermay be branched or unbranched, substituted or unsubstituted, andchirally pure or a racemic mixture. Most preferably, such alkyl linkershave from about 2 to about 18 carbon atoms. In some preferredembodiments such alkyl linkers have from about 3 to about 9 carbonatoms. Such alkyl linkers include polyethyleneglycol linkers[—O—CH2-CH2-]_(n) (n=2-9). In some preferred embodiments, such alkyllinkers may include peptides or amino acids.

[0068] Preferred immunostimulatory moieties according to the inventionfurther include DNA isoforms, including without limitationβ-L-deoxynucleosides and alpha-deoxynucleosides. Preferredimmunostimulatory moieties according to the invention further includenucleosides having unnatural internucleoside linkage positions,including without limitation 2′-5′,2′-2′,3′-3′ and 5′-5′ linkages.

[0069] Preferred immunostimulatory moieties according to the inventionfurther include nucleosides having modified heterocyclic bases,including without limitation 5-hydroxydeoxycytidine,5-hydroxymethyldeoxycytidine, N4-alkyldeoxycytidine, preferablyN4-ethyldeoxycytidine, 4-thiouridine, 6-thiodeoxyguanosine,7-deaza-guanosine, and deoxyribonucleosides of nitropyrrole,C5-propynylpyrimidine, and diaminopurine, including without limitation2,6-diaminopurine.

[0070] By way of specific illustration and not by way of limitation, forexample, in the immunostimulatory domain described earlier 5′ - - -X1-X2-Y-Z-X3-X4 - - - 3′

[0071] a nucleoside methylphosphonate at position X3 or X4 is animmunostimulatory moiety, a substituted or unsubstituted alkyl linker atposition X1 is an immunostimulatory moiety, and a β-L-deoxynucleoside atposition X1 is an immunostimulatory moiety. See Table 1 below forrepresentative positions and structures of immunostimulatory moietieswithin the immunostimulatory domain. TABLE 1 Position TYPICALIMMUNOSTIMULATORY MOIETIES X1 C3-alkyl linker,2-aminobutyl-1,3-propanediol linker (amino linker), β-L-deoxynucleosideX2 2-aminobutyl-1,3-propanediol linker X3 nucleoside methylphosphonateX4 nucleoside methylphosphonate, 2′-O-methyl-ribonucleoside

[0072] In some embodiments, the immunostimulatory oligonucleotidefurther comprises a potentiation domain

[0073] A “potentiation domain” is a region of an immunostimulatoryoligonucleotide analog, other than the immunostimulatory domain, thatcauses the oligonucleotide to be more immunostimulatory if it containsthe potentiation domain than the oligonucleotide would be in the absenceof the potentiation domain. The potentiation domain can be upstream ordownstream relative to the immunostimulatory domain. The term “upstream”is used to refer to positions on the 5′ side of the immunostimulatorydinucleotide or dinucleotide analog (Y-Z). The term “downstream” is usedto refer to positions on the 3′ side of Y-Z.

[0074] For example, an immunostimulatory oligonucleotide analog couldhave the structure 5′-U9-U8-U7-U6-U5-U4-U3-U2-U1-X1-X2-Y-Z-X3-X4-N-N-N-3′

[0075] wherein U9-U1 represents an upstream potentiation domain, whereineach U independently represents the same or a different nucleosideimmunostimulatory moiety, N represents any nucleoside and X1-X4, Y and Zare as before.

[0076] Alternatively, an immunostimulatory oligonucleotide analog couldhave the structure 5′-N-N-X1-X2-Y-Z-X3-X4-D1-D2-D3-D4-D5-D6-D7-D8-3′

[0077] wherein D1-D8 represents a downstream potentiation domain,wherein each D independently represents the same or a differentnucleoside or immunostimulatory moiety, and all other symbols are asdescribed above.

[0078] In these configurations, an immunostimulatory moiety at U6 wouldbe six positions upstream from the immunostimulatory dinucleotide ordinucleotide analog and an immunostimulatory moiety at D4 would be fourpositions downstream from the immunostimulatory dinucleotide ordinucleotide analog. The term “position” is used rather than“nucleoside”, because any of the U or D positions can represent animmunostimulatory moiety which may or may not be a nucleoside ornucleoside analog. Of course, oligonucleotide analogs can be constructedhaving both upstream and downstream potentiation domains.

[0079] Table 2 shows representative positions and structures ofimmunostimulatory moieties within an immunostimulatory oligonucleotidehaving an upstream potentiation domain. See FIG. 7 for definitions ofSpacer 9 and Spacer 18 as referred to in Tables 2 and 3. TABLE 2Position TYPICAL IMMUNOSTIMULATORY MOIETY X22-aminobutyl-1,3-propanediol linker X1 C3-linker,2-aminobutyl-1,3-propanediol linker, β-L- deoxynucleoside U11′,2′-dideoxyribose, C3-linker, 2′-O-methyl-ribonucleoside U21′,2′-dideoxyribose, C3-linker, Spacer 18, 3′-deoxynucleoside,nucleoside methylphosphonate, β-L-deoxynucleoside, 2′-O-propargyl-ribonucleoside U3 1′,2′-dideoxyribose, C3-linker, Spacer 9,Spacer 18, nucleoside methylphosphonate, 2′-5′ linkage U2 + U31′,2′-dideoxyribose, C3-linker,, β-L-deoxynucleoside U3 + U4 nucleosidemethylphosphonate, 2′-O-methoxyethyl- ribonucleoside U5 + U61′,2′-dideoxyribose, C3-linker X1 + U3 1′,2′-dideoxyribose

[0080] Table 3-shows representative positions and structures ofimmunostimulatory moieties within an immunostimulatory oligonucleotidehaving a downstream potentiation domain. TABLE 3 Position TYPICALIMMUNOSTIMULATORY MOIETY X3 nucleoside methylphosphonate X4 nucleosidemethylphosphonate, 2′-O-methyl-ribonucleoside D1 1′,2′-dideoxyribose,nucleoside methylphosphonate D2 1′,2′-dideoxyribose, C3-linker, Spacer9, Spacer 18, 2- aminobutyl-1,3-propanediol-linker, nucleosidemethylphosphonate, β-L-deoxynucleoside D3 3′-deoxynucleoside,2′-O-propargyl-ribonucleoside, 2′-5′-linkage D2 + D31′,2′-dideoxyribose, β-L-deoxynucleoside

[0081] In another embodiment of the invention, the oligonucleotideaccording to the invention has one or two accessible 5′ ends. Thepresent inventors have discovered that immunostimulatory moieties in theregion 5′ to the immunostimulatory dinucleotide have a greater impact onimmunostimulatory activity than do similar substitutions in the region3′ to the immunostimulatory dinucleotide. This observation suggests thatthe 5′-flanking region of CpG-PS-oligos plays an important role inimmunostimulatory activity. Moreover, the inventors have discovered thatcompounds having two oligonucleotide units attached by way of a 3′-5′ or3′-3′ linkage have greater immunostimulatory activity than do compoundsin which the two oligonucleotide units are attached by way of a 5′-5′linkage. In some preferred embodiments, therefore, the immunostimulatoryoligonucleotide according to the invention comprises a 3′-3′ linkage. Insome such embodiments, the oligonucleotides have one or two accessible5′ ends.

[0082] In a second aspect, the invention provides methods for modulatingthe immunostimulatory effect of an immunostimulatory oligonucleotide. Insome embodiments, the method comprises introducing into theimmunostimulatory domain a dinucleotide analog that includes anon-naturally occurring pyrimidine base, as described above for thefirst aspect of the invention. In some embodiments, the method comprisesintroducing into the immunostimulatory domain and/or potentiation domainan immunostimulatory moiety at a specified position, as described above.In some embodiments, the method comprises introducing into theoligonucleotide a 3′-3′ linkage.

[0083] For purposes of the invention, “introducing an immunostimulatorymoiety” at a specified position simply means synthesizing anoligonucleotide that has an immunostimulatory moiety at the specifiedposition. For example, “introducing an immunostimulatory moiety intoposition U6” simply means synthesizing an oligonucleotide that has animmunostimulatory moiety at such a position, with reference to, e.g.,the following structure:5′-U9-U8-U7-U6-U5-U4-U3-U2-U1-X1-X2-Y-Z-X3-X4-D1-D2-D3-3′.

[0084] Preferably, the methods according to this aspect of the inventioninclude introducing an immunostimulatory moiety at a position in theimmunostimulatory domain or in an upstream or downstream potentiationdomain according to the preferred substitution patterns described inTables 1-3.

[0085] The methods according to this aspect of the invention can beconveniently carried out using any of the well-known synthesistechniques by simply using an appropriate immunomodulatory moietymonomer synthon in the synthesis process in an appropriate cycle toobtain the desired position. Preferred monomers includephosphoramidites, phosphotriesters and H-phosphonates. PS-oligos arereadily synthesized, e.g., using β-cyanoethylphosphoramidite chemistryon CPG solid support using appropriate phosphoramidites, deprotected asrequired, purified by C₁₈ reverse phase HPLC, dialyzed against distilledwater and lyophilized. The purity of each PS-oligo is readily determinedby CGE and the molecular weight can be confirmed by MALDI-TOF massspectral analysis.

[0086] In a third aspect, the invention provides methods for generatingan immune response in a patient, such methods comprising administeringto the patient an oligonucleotide analog immunostimulatory compoundaccording to the invention.

[0087] In the methods according to this aspect of the invention,preferably, administration of compounds is parenteral, oral, sublingual,transdermal, topical, intranasal, intratracheal, intravaginal, orintrarectal. Administration of the therapeutic compositions can becarried out using known procedures at dosages and for periods of timeeffective to reduce symptoms or surrogate markers of the disease. Whenadministered systemically, the therapeutic composition is preferablyadministered at a sufficient dosage to attain a blood level ofoligonucleotide from about 0.001 micromolar to about 10 micromolar. Forlocalized administration, much lower concentrations than this may beeffective, and much higher concentrations may be tolerated. Preferably,a total dosage of oligonucleotide will range from about 0.1 mgoligonucleotide per patient per day to about 40 mg oligonucleotide perkg body weight per day. It may be desirable to administersimultaneously, or sequentially a therapeutically effective amount ofone or more of the therapeutic compositions of the invention to anindividual as a single treatment episode. In some instances, dosagesbelow the above-defined ranges may still provide efficacy. In apreferred embodiment, after the composition of matter is administered,one or more measurement is taken of biological effects selected from thegroup consisting of complement activation, mitogenesis and inhibition ofthrombin clot formation.

[0088] In certain preferred embodiments, compounds according to theinvention are administered in combination with antibiotics, antigens,allergens, vaccines, antibodies, cytotoxic agents, antisenseoligonucleotides, gene therapy vectors, DNA vaccines and/or adjuvants toenhance the specificity or magnitude of the immune response. Either thecompound or the vaccine, or both may optionally be linked to animmunogenic protein, such as keyhole limpet hemocyanin, cholera toxin Bsubunit, or any other immunogenic carrier protein. Any of a plethora ofadjuvants may be used, including, without limitation, Freund's completeadjuvant, monophosphoryl lipid A (MPL), saponins, including QS-21, alum,and combinations thereof. Certain preferred embodiments of the methodsaccording to the invention induce cytokines by administration ofimmunostimulatory oligonucleotide compounds. In certain embodiments theimmunostimulatory oligonucleotide compounds are conjugated to anantigen, hapten, or vaccine. As discussed above, the present inventorshave discovered that an accessible 5′ end is important to the activityof certain immunostimulatory oligonucleotide compounds. Accordingly, foroptimum immunostimulatory activity, the oligonucleotide preferably isconjugated to an antigen or vaccine by means of the 3′-end ofoligonucleotide compound.

[0089] For purposes of this aspect “in combination with” means in thecourse of treating the same disease in the same patient, and includesadministering the oligonucleotide and/or the vaccine and/or the adjuvantin any order, including simultaneous administration, as well astemporally spaced order of up to several days apart. Such combinationtreatment may also include more than a single administration of theoligonucleotide, and/or independently the vaccine, and/or independentlythe adjuvant. The administration of the oligonucleotide and/or vaccineand/or adjuvant may be by the same or different routes.

[0090] The method according to this aspect of the invention is usefulfor model studies of the immune system, and is further useful for thetherapeutic treatment of human or animal disease.

[0091] In a fourth aspect, the invention provides methods fortherapeutically treating a patient having disease caused by a pathogen,such methods comprising administering to the patient an oligonucleotideanalog immunostimulatory compound according to the invention.Administration is carried out as described for the third aspect of theinvention.

[0092] In a fifth aspect, the invention provides methods for treating acancer patient, such methods comprising administering to the patient anoligonucleotide analog immunostimulatory compound according to theinvention. Administration is carried out as described for the thirdaspect of the invention.

[0093] In a sixth aspect, the invention provides methods for treatingautoimmune disorders, such as autoimmune asthma, such methods comprisingadministering to the patient an oligonucleotide analog immunostimulatorycompound according to the invention. Administration is carried out asdescribed for the third aspect of the invention.

[0094] In a seventh aspect, the invention provides methods for treatingairway inflammation or allergies, such methods comprising administeringto the patient an oligonucleotide analog immunostimulatory compoundaccording to the invention. Administration is carried out as describedfor the third aspect of the invention.

[0095] The following examples are intended to further illustrate certainpreferred embodiments of the invention, and are not intended to limitthe scope of the invention.

EXAMPLES Example 1 Synthesis of Oligonucleotides ContainingImmunomodulatory Moieties

[0096] Oligonucleotides were synthesized on a 1 micromolar scale usingan automated DNA synthesizer (Expedite 8909, PerSeptive Biosystems,Foster City, Calif.). Standard deoxynucleoside phosphoramidites areobtained from PerSeptive Biosystems. 1′,2′-dideoxyribosephosphoramidite, propyl-1-phosphoramidite,2′-deoxy-5-nitroindole-ribofuranosyl phosphoramidite, 2′-deoxy-uridinephosphoramidite, 2′-deoxy-P phosphoramidite, 2′-deoxy-2-aminopurinephosphoramidite, 2′-deoxy-nebularine phosphoramidite,2′-deoxy-7-deazaguanosine phosphoramidite, 2′-deoxy-4-thiouridinephosphoramidite, 2′-deoxy-isoguanosine phosphoramidite,2′-deoxy-5-methylisocytosine phosphoramidite, 2′-deoxy-4-thiothymidinephosphoramidite, 2′-deoxy-K-phosphoramidite, 2′-deoxy-2-aminoadenosinephosphoramidite, 2′-deoxy-N-4-ethyl-cytosine phosphoramidite,2′-deoxy-6-thioguanosine phosphoramidite, 2′-deoxy-7-deaza-xanthosinephosphoramidite, 2′-deoxy-8-bromoguanosine phosphoramidite,2′-deoxy-8-oxoguanosine phosphoramidite, 2′-deoxy-5-hydroxycytosinephosphoramidite, arabino-cytosine phosphoramidite and2′-deoxy-5-propynecytosine phosphoramidite were obtained from GlenResearch (Sterling, Va.). 2′-Deoxy-inosine phosphoramidite were obtainedfrom ChemGenes (Ashland, Md.).

[0097] Normal coupling cycles or a coupling cycle recommended by thephosphoramidite manufacturer were used for all phosphoramidites.Beaucage reagent was used as an oxidant to obtain phosphorothioatemodification. After synthesis, oligonucleotides were deprotected byincubating CPG-bound oligonucleotide with concentrated ammoniumhydroxide solution for 1.5-2 hours at room temperature and thenincubating the ammonium hydroxide supernatant for 12 hours at 55 degreesC. or as recommended by phosphoramidite manufacturer. The ammoniumhydroxide solution was evaporated to dryness in a speed-vac and5′-DMTr-oligonucleotides were purified by HPLC on a C18 reverse-phasematrix using a solvent system of 0.1 M ammonium acetate and 1:5 ratio0.1 M ammonium acetate in acetonitrile. Then the oligonucleotides weretreated with 80% acetic acid to remove the DMTr group, converted tosodium form and desalted by dialysis against double distilled water.Oligonucleotides were filtered through 0.4 μg filters, lyophilized andredissolved in double distilled water. Characterization was achieved bydenaturing PAGE and MALDI-TOF mass spectrometry.

Example 2 Synthesis of CpG-PS-Oligos Containing Cytosine Analogs

[0098] Following the procedures outlined in Example 1, the followingoligonucleotides were synthesized: Oligo # Sequence (5′---> 3′) andModification^(a) 1 d(CTATCTGACGTTCTCTGT) 2 d(CTATCTGAC*GTTCTCTGT) 3d(CTATCTGACC*TTCTCTGT) 4 d(CTATCTGAC*GTTCTCTGT) 5 d(CTATCTGACC*TTCTCTGT)

[0099] The oligonucleotides were characterized by CGE and MALDI-TOF massspectrometry (Brucker Proflex III MALDI-TOF mass spectrometer with 337nm N2 laser). Molecular weights observed and calculated (shown inparentheses) for each oligonucleotide are as follows: Oligo 1, 5704(5704.8); Oligo 2, 5720 (5720.8); Oligo 3, 5681 (5680.7); Oligo 4, 5733(5733); Oligo 5, 5694 (5693).

Example 3 Analysis of Spleen Weights in Treated Mice

[0100] Female BALB/c mice (4-5 weeks, 19-21 g, Charles River,Wilmington, Mass.) were used in the study. The animals were fed withcommercial diet and water ad lib. The animals were injectedintraperitoneally with 5 or 10 mg/kg dose of immunostimulatoryoligonucleotide compound dissolved in sterile PBS. One group of micereceived PBS alone to serve as a control (PBS). Four animals were usedfor each immunostimulatory oligonucleotide compound. Mice weresacrificed 72 h later, spleens were harvested and weighed.

Example 4 Analysis of Immunostimulatory Oligonucleotide Compounds inMouse Lymphocyte Proliferation Assay

[0101] Spleens from CD-1, BALB/c, C57BL/6 mouse (4-8 weeks) were used assource of lymphocytes. Single cell suspensions were prepared by gentlymincing with the frosted ends of glass slides. Cells were then culturedin RPMI complete medium [RPMI medium supplemented with 10% fetal bovineserum (FBS) (heat-inactivated at 56° C. for 30 min), 50 μM2-mercaptoethanol, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mML-glutamine]. The cells were then plated in 96-well dishes at a densityof 10⁶ cells/mL in a final volume of 100 μL. Immunostimulatoryoligonucleotide compounds or LPS (lipopolysaccharide) were added to thecell culture in 10 μL of TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA).The cells were then set to culture at 37° C. After 44 h, 1 μCi³H-uridine (Amersham, Arlington Heights, Ill.) was added to the culturein 20 μL of RPMI medium, and the cells were pulse-labeled for another 4h. The cells were harvested by automatic cell harvester (Skatron,Sterling, Va.), and the filters were counted by a scintillation counter.The experiments were performed in triplicate.

Example 5 Lymphocyte Proliferatory Activity of CpG-PS-Oligos ContainingCytosine Analogs

[0102] The immunostimulatory activity of CpG-PS-oligos 1-5 (Example 4)was studied using a BALB/c mouse lymphocyte proliferation assay. Inbrief, mouse spleen cells were cultured and incubated with CpG-PS-oligosat 0.1, 0.3, 1.0 and 3.0 μg/mL concentration for 48 hr and cellproliferation was measured by ³H-uridine incorporation.

[0103]FIG. 23 shows the dose-dependent cell proliferatory activity ofoligos 1-5 in mouse lymphocyte cultures. At a dose of 3.0 μg/mL, oligo1, with natural cytidine, showed a proliferation index of 29.5±2.1.Oligo 2, in which the cytosine base of the deoxycytidine of theCpG-motif is replaced with a 5-hydroxycytosine, also showeddose-dependent lymphocyte proliferation. A proliferation index of23.7±2.9 at 3.0 μg/mL dose was observed for oligo 2. PS-Oligo 4, whichcontained N4-ethyl-cytosine in place of the cytosine base in theCpG-motif, also showed dose-dependent cell-proliferation activity. Theproliferation index of 18.7±1.6 observed for oligo 4 at a dose of 3μg/mL suggests that the presence of a bulky hydrophobic substitution onthe 4-amino group of cytosine in a CpG-motif slightly impedesimmunostimulatory activity.

[0104] Oligo 3, in which 5-hydroxydeoxycytidine was placed in thedeoxyguanosine position instead of the deoxycytidine position of theCpG-motif, showed a proliferation index that was similar to thatobserved for media control (FIG. 23). Similarly, the control Oligo 5 inwhich deoxyguanosine in the CpG-motif was substituted withN4-ethyldeoxycytidine, showed cell proliferation similar to that ofmedia control.

[0105] Other oligos, in which cytosine base in the CpG-motif wasreplaced with 5-methyl-deoxycytosine (2; see FIG. 28),5-methyl-deoxyisocytosine (3), deoxyuridine (5), or deoxy-P-base (7)showed no or insignificant cell proliferatory activity in the same assaysystem. These results suggest that (i) cell proliferatory activity ismaintained when the cytosine base of the CpG motif is replaced with5-hydroxycytosine or N4-ethylcytosine (Oligos 2 and 4, respectively),but (ii) substitution of the guanine base with these cytosine analogsresults in a loss of cell proliferatory activity.

Example 6 Splenomegaly in Mice Induced by CpG-PS-Oligos ContainingCytosine Analogs

[0106] To confirm the in vitro effects of CpG-PS-oligos, Oligos 1, 2,and 4 (from Example 4) were injected intraperitoneally (ip) to BALB/cmice at a dose of 10 mg/kg and the change in spleen weight was measuredas an indicator of the level of immunostimulatory activity of eachPS-oligo. The change in spleen weight as a result of treatment withCpG-PS-oligos is presented in FIG. 24. Female BALB/c mice (4-6 weeks,19-21 gm) were divided in to different groups with four mice in eachgroup. Oligonucleotides were dissolved in sterile PBS and administeredintraperitoneally to mice at a dose of 10 mg/kg. After 72 hr, mice weresacrificed and spleens were harvested and weighed. Each circlerepresents the spleen weight of an individual mouse and the + representsthe mean spleen weight for each group.

[0107] Oligo 1, which has natural deoxycytidine in the CpG-motif, showedabout 45% increase in spleen weight at a dose of 10 mg/kg, compared withthe control group of mice that received PBS. Oligo 2, which has a5-hydroxycytosine in place of the cytosine base in the CpG-motif, showedabout 35% increase in spleen weight at the same dose. Oligo 4, which hasN4-ethylcytosine in place of the cytosine base in the CpG-motif, showedabout 34% increase in spleen weight at the same dose compared to thecontrol group. These data confirm the results observed in lymphocyteproliferation assays for these oligos containing modified cytidineanalogs in place of deoxycytidine in the CpG-motif.

Example 7 Structure-Activity Relationships of C*pG-PS-Oligos

[0108] The presence of a methyl group at the 5-position of cytosine(5-methyl-deoxycytosine, 2 (FIG. 28)) in a CpG-motif completelyabolishes CpG related immunostimulatory effects of CpG-PS-oligos. Basedon the results observed in in vitro and in vivo experiments we haveconstructed structure-activity relationships for the PS-oligoscontaining cytosine analogs.

[0109] The replacement of the cytosine base (1) in the CpG-motif with5-methyl-isocytosine (3) resulted in complete loss of immunostimulatoryactivity, as is the case with 5-methylcytosine (2), which could be as aresult of switching the keto and amino groups at the 2 and 4-positions,respectively, and/or placing a hydrophobic methyl group at the5-position of cytosine.

[0110] Oligo 2, containing a hydrophilic hydroxy substitution at the5-position of the cytosine in the CpG-motif, showed immunostimulatoryactivity similar to that of oligo 1, which contains the natural cytosinebase. This observation suggests that bulky hydrophilic groups are bettertolerated than are hydrophobic groups at the 5-position of cytosine forimmunostimulatory activity of CpG-PS-oligos. Perhaps the binding pocketfor the CpG-oligos on receptor is hydrophilic in nature and can notaccommodate a hydrophobic group at the 5-position of cytosine.

[0111] When the cytosine base in the CpG-motif is replaced with uracil(5 (see FIG. 28)), in which keto groups are present at both the 2 and4-positions, no immunostimulatory activity was observed, suggesting thata hydrogen bond donor amino group at the 4-position of cytosine iscritical for immunostimulatory activity. When a large hydrophobic ethylgroup is placed on 4-amino group of cytosine in a CpG-motif, reducedlymphocyte proliferation and a slightly reduced increase in spleenweight in mice were observed, suggesting that a bulky ethyl group atthis position does not interfere with binding of the CpG-PS-oligo to thereceptor factors responsible for immunostimulatory activity. In spite ofthe ethyl substitution, the 4-amino group of N4-ethylcytosine (6) canparticipate in hydrogen bond formation with an acceptor. The modifiedpyrimidine base dP, in which the nitrogen group located at the4-position involved in ring structure formation with the 5-position, andwhich does not have a hydrogen bond donor amino group at the 4-position,had no mouse lymphocyte proliferation activity in cultures, suggestingthat the 4-amino group of cytosine in a CpG-motif is critical forimmunostimulatory activity.

[0112] In conclusion, the results presented here show that thefunctional groups at 2, 3, and 4 positions of the cytosine are importantfor CpG-related immunostimulatory activity. A hydrophobic substitutionat the 5-position of cytosine completely suppresses immunostimulatoryactivity of a CpG-oligo, while a hydrophilic group at this position istolerated well. In addition, the immunostimulatory activity ofCpG-PS-oligos containing 5-hydroxycytosine or N4-ethylcytosine in placeof cytosine in the CpG-motif can be modulated significantly byincorporating appropriate chemical modifications in the 5′-flankingsequence, suggesting that these cytosine analogs in a CpG-motif arerecognized as part of an immunostimulatory motif.

Example 8 Synthesis of End-Blocked CpG-PS Oligonucleotides

[0113] The CpG-PS-oligos shown in FIG. 17 were synthesized using anautomated synthesizer and phosphoramidite approach. Oligo 1 (16-mer) wassynthesized using nucleoside-5′-β-cyanoethylphosphoramidites. Oligo 2, a32-mer, was synthesized usingnucleoside-3′-β-cyanoethylphosphor-amidites and controlled pore glasssupport (CPG-solid support) with a 3′-linked nucleoside in which 16-mersequence of Oligo 1 was repeated twice; therefore, Oligo 2 had two16-mers (Oligo 1) linked by a normal 3′-5′-linkage. Oligo 3, a 32-mer,was synthesized with two 16-mers (Oligo 1) linked by a 5′-5′-linkage, soOligo 3 had two 3′-ends and no 5′-end. Synthesis of Oligo 3 was carriedout in two steps: the first 16-mer was synthesized usingnucleoside-3′-β-cyano-ethylphosphoramidites and solid support with a3′-linked nucleoside, and then synthesis of the second 16-mer segmentwas continued using nucleoside-5′-β-cyano-ethylphosphoramidites. Oligo4, a 32-mer, comprised two 16-mers (Oligo 1) linked by a 3′-3′-linkage,so Oligo 4 had two 5′-ends and no 3′-end. Synthesis of Oligo 4 wascarried out in two steps: the first 16-mer was synthesized usingnucleoside-5′-β-cyano-ethylphosphoramidites and solid support with a5′-linked nucleoside, and the synthesis of the second 16-mer segment wascontinued using nucleoside-3′-β-cyanoethyl-phosphoramidites. Synthesisof Oligos 5-8 was carried out by using the samenucleoside-β-cyanoethylphosphoramidites as for Oligos 1-4, respectively.At the end of the synthesis, Oligos 1-8 were deprotected withconcentrated ammonia solution, purified by reversed phase HPLC,detritylated, desalted and dialyzed. The purity of each PS-oligo waschecked by CGE and the molecular weight was confirmed by MALDI-TOF massspectral analysis (Table 1). The sequence integrity and directionalityof 5′-CpG motif in Oligos 1-8 were confirmed by recording meltingtemperatures (T_(m)s) of the duplexes with their respective DNAcomplementary strands (5′-AAGGTCGAGCGTTCTC-3′ for Oligos 1-4, and5′-ATGGCGCACGCTGGGAGA-3′ for Oligos 5-8). The T_(m)s of these duplexeswere 53.9±0.9° C. (Oligos 1-4), 61.8° C. (Oligo 5), and 58.8±0.6° C.(Oligos 6-8) (note that Oligo 5 was a 18-mer and Oligos 6-8 were 32-mersbut not 36-mers).

Example 9 Mouse Spleen Lymphocyte Proliferatory Activity of End-BlockedCpG-PS Oligonucleotides

[0114] Immunostimulatory activity of the end-blocked CpG-PS-oligos ofExample 8 was studied initially in a lymphocyte proliferation assay.Typically, mouse (Balb-C) spleen lymphocytes were cultured withCpG-PS-oligos at concentrations of 0.1, 1.0, and 10.0 μg/ml for 48 h andcell proliferation was determined by ³H-uridine incorporation, asdescribed in Example 3. Results are shown in FIG. 17

[0115] Oligo 1 induced a dose-dependent effect on cell proliferation; ata concentration of 10 μg/ml (˜2.0 μM), the proliferation index was5.0±0.32. Oligo 2, which consisted of two units of Oligo 1 linked by a3′-5′-linkage, had a proliferation index of 5.8±0.28 at the same dose(˜1.0 μM). Oligo 3, which consisted of two units of Oligo 1 linked by a5′-5′-linkage, had a proliferation index of 2.0±0.26, reflecting asignificantly lower immunostimulatory activity than observed with Oligos1 and 2. Oligo 4, which consisted of two units of Oligo 1 linked by a3′-3′-linkage, had a proliferation index of 7.2±0.5, reflecting agreater immunostimulatory activity than observed with Oligos 1 and 2.

[0116] Similar results were obtained with Oligos 5-8. Oligo 5 had aproliferation index of 3.9±0.12. Oligos 6-8, in which two units of Oligo5 are linked by a 3′-5′-linkage (Oligo 6), 5′-5′-linkage (Oligo 7), and3′-3′-linkage (Oligo 8) had proliferation indices of 4.9±0.2, 1.74±0.21,and 7.7±0.82, respectively. Comparison of the results obtained withOligos 6-8 show that Oligos 6 and 8, in which two Oligo 5 sequences werelinked by a 3′-5′-linkage or a 3′-3′-linkage had greaterimmunostimulatory activity, while Oligo 7, in which two Oligo 5 werelinked by a 5′-5′-linkage had significant less immunostimulatoryactivity, than did Oligo 5.

[0117] Based on lymphocyte proliferation results of Oligos 1-8, it isclear that when oligos are linked through their 5′-ends, there is asignificant loss of immunostimulatory activity, while if they are linkedthrough their 3′-ends, there is an increase in immunostimulatoryactivity. It is important to note that 3′-3′-linked oligos have shownsubstantially greater stability towards degradation by exonucleases thanthe oligos that contained a free 3′-end, which could also result inincreased immunostimulatory activity. The lower immunostimulatoryactivity of Oligos 3 and 7, in which the 5′-end of oligos is blocked,suggests that accessibility to 5′-end of oligo is essential forimmunostimulatory activity of CpG-PS-oligos.

Example 10 Splenomegaly in Mice Induced by End-Blocked CpG-PSOligonucleotides

[0118] To confirm the immunostimulatory activity of Oligos 1-8 (Example8) in vivo, a dose of 5 mg/kg of oligonucleotides was injectedintraperitoneally to Balb-C mice. The mice were sacrificed 72 hourspost-administration, spleens were removed, blotted to dryness, andweighed. Change in spleen weight in treated and untreated mice was usedas a parameter for immunostimulatory activity.

[0119] Administration 5 mg/kg dose of Oligo 1 caused about 40% increasein spleen weight compared with the control mice that received PBS.Administration of Oligos 2 and 4 also caused about 50% increase inspleen weight. Administration of Oligo 3 caused no difference in spleenweight compared with control mice. These results further support theobservation that Oligo 3, in which 5′-end was blocked, had significantlyless immunostimulatory activity compared to oligos that had accessible5′-end. These results were also confirmed with the administration ofOligos 5-8. Administration of Oligos 5, 6, and 8 caused about 40-50%increase in spleen weight, whereas no change in spleen weight wasobserved following the administration of Oligo 7.

[0120] The above results suggest that the immunostimulatory activity ofPS-oligos containing a CpG motif is significantly minimized if the5′-end of the oligo is not accessible. This loss in immunostimulatoryactivity of Oligos 3 and 7 cannot be explained based on nucleasestability, as both oligos have two 3′-ends and are not more susceptibleto 3′-exonuclease degradation than are Oligos 1, 2, 5, and 6, which haveone 3′-end. PS-Oligos 4 and 8, which have their 3′-ends blocked and arevery stable to degradation by exonucleases, showed similarimmunostimulatory activity. Oligos 4 and 8 may show sustainedimmunostimulatory activity due to their increased in vivo stability,which is not evident in the present study as mice were sacrificed atonly 72 hours after administration. Studies are in progress in whichmice will be sacrificed at times later than 72 hours afteradministration.

[0121] The results described here are intriguing and suggest that the5′-end of CpG-PS-oligos is critical for immunostimulatory activity. Asdiscussed here, we have shown that substitution of deoxynucleosides in5′-flanking regions by modified 2′- or 3′-substituted ribonucleosidesresulted in increased immunostimulatory activity. In addition,substitution of deoxynucleosides immediately upstream (5′-end) to theCpG motif caused a significant suppression and substitution ofdeoxynucleosides immediately downstream (3′-end) to the CpG motif had noeffect on immunostimulatory activity. Taken together, these resultssuggest that the enzyme/receptor responsible for the immunestimulationrecognizes the CpG motif in oligos from the 5′-end and requiresaccessibility to the 5′-end.

[0122] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

1 112 1 18 DNA Artificial Sequence synthesis of CpG-PS-oligos containingcytosine analogs 1 ctatctgacg ttctctgt 18 2 18 DNA Artificial Sequencesynthesis of CpG-PS-oligos containing cytosine analogs 2 ctatctgacgttctctgt 18 3 18 DNA Artificial Sequence synthesis of CpG-PS-oligoscontaining cytosine analogs 3 ctatctgacc ttctctgt 18 4 18 DNA ArtificialSequence synthesis of CpG-PS-oligos containing cytosine analogs 4ctatctgacg ttctctgt 18 5 18 DNA Artificial Sequence synthesis ofCpG-PS-oligos containing cytosine analogs 5 ctatctgacc ttctctgt 18 6 16DNA Artificial Sequence synthesis of end-blocked CpG-PS modifiedoligodeoxynucleotide phosphorothioate 6 aaggtcgagc gttctc 16 7 18 DNAArtificial Sequence synthesis of end-blocked CpG-PS modifiedoligodeoxynucleotide phosphorothioate 7 atggcgcacg ctgggaga 18 8 18 DNAArtificial Sequence oligodeoxynucleotide phosphorothioate 8 cctactagcgttctcatc 18 9 18 DNA Artificial Sequence oligodeoxynucleotidephosphorothioate 9 cctactagcg ttctcatc 18 10 18 DNA Artificial Sequencemodified oligodeoxynucleotide phosphorothioate 10 cctactagcg ttctcatc 1811 18 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 11 cctactagcg ttctcatc 18 12 18 DNA Artificial Sequencemodified oligodeoxynucleotide phosphorothioate 12 cctactagcg ttctcatc 1813 18 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 13 cctactagcg ttctcatc 18 14 18 DNA Artificial Sequencemodified oligodeoxynucleotide phosphorothioate 14 cctactagcg ttctcatc 1815 18 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 15 cctactagcc ttctcatc 18 16 18 DNA Artificial Sequencemodified oligodeoxynucleotide phosphorothioate 16 cctactagcg ttctcatc 1817 18 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 17 cctactagcg ttctcatc 18 18 18 DNA Artificial Sequencemodified oligodeoxynucleotide phosphorothioate 18 cctactagcg ttctcatc 1819 18 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 19 cctactagcg ttctcatc 18 20 18 DNA Artificial Sequencemodified oligodeoxynucleotide phosphorothioate 20 cctactagcg ttctcatc 1821 18 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 21 cctactagcg ttctcatc 18 22 18 DNA Artificial Sequencemodified oligodeoxynucleotide phosphorothioate 22 cctactagcg ttctcatc 1823 18 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 23 cctactagcg ttctcatc 18 24 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 24 cctactagcgttctcatc 18 25 18 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 25 cctactagcg ttctcatc 18 26 18DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 26 cctactagcg ttctcatc 18 27 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 27 cctactagcgttctcatc 18 28 18 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 28 cctactagcg ttctcatc 18 29 18DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 29 cctactagcg ttctcatc 18 30 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 30 cctactagcgttctcatc 18 31 18 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 31 cctactagcg ttctcatc 18 32 18DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 32 ctatctgacg ttctctgt 18 33 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 33 ctatctgacgttctctgt 18 34 18 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 34 ctatctgacg ttctctgt 18 35 18DNA Artificial Sequence modified oligodeoxynucleotide phosphorothioate35 ctatctgacg ttctctgt 18 36 18 DNA Artificial Sequence modifiedoligodeoxynucleotide phosphorothioate 36 ctatctgacg ttctctgt 18 37 18DNA Artificial Sequence modified oligodeoxynucleotide phosphorothioate37 ctatctgacg ttctctgt 18 38 18 DNA Artificial Sequence modifiedoligodeoxynucleotide phosphorothioate 38 ctatctgacg ttctctgt 18 39 18DNA Artificial Sequence modified oligodeoxynucleotide phosphorothioate39 cctactagcg ttctcatc 18 40 18 DNA Artificial Sequence modifiedoligodeoxynucleotide phosphorothioate 40 cctactagcg ttctcatc 18 41 18DNA Artificial Sequence modified oligodeoxynucleotide phosphorothioate41 cctactagcg ttctcatc 18 42 18 DNA Artificial Sequence modifiedoligodeoxynucleotide phosphorothioate 42 cctactagcg ttctcatc 18 43 18DNA Artificial Sequence modified oligodeoxynucleotide phosphorothioate43 ctatctgacg ttctctgt 18 44 18 DNA Artificial Sequence modifiedoligodeoxynucleotide phosphorothioate 44 ctatctgacg ttctctgt 18 45 18DNA Artificial Sequence modified oligodeoxynucleotide phosphorothioate45 ctatctgacg ttctctgt 18 46 18 DNA Artificial Sequence modifiedoligodeoxynucleotide phosphorothioate 46 ctatctgacg ttctctgt 18 47 18DNA Artificial Sequence modified oligodeoxynucleotide phosphorothioate47 ctatctgacg ttctctgt 18 48 18 DNA Artificial Sequence modified linkageof oligodeoxynucleotide phosphorothioate 48 ctatctgacg ttctctgt 18 49 18DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 49 ctatctgacg ttctctgt 18 50 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 50 ctatctgacgttctctgt 18 51 18 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 51 ctatctgacg ttctctgt 18 52 18DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 52 cctactagcg ttctcatc 18 53 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 53 cctactagcgttctcatc 18 54 18 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 54 cctactagcg ttctcatc 18 55 18DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 55 cctactagcg ttctcatc 18 56 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 56 cctactagcgttctcatc 18 57 18 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 57 ctatctgacg ttctctgt 18 58 18DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 58 ctatctgacg ttctctgt 18 59 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 59 ctatctgacgttctctgt 18 60 18 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 60 ctatctgacg ttctctgt 18 61 18DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 61 ctatctgacg ttctctgt 18 62 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 62 ctatctgacgttctctgt 18 63 18 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 63 ctatctgacg ttctctgt 18 64 18DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 64 ctatctgacg ttctctgt 18 65 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 65 ctatctgacgttctctgt 18 66 18 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 66 ctatctgacg ttctctgt 18 67 18DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 67 ctatctgacg ttctctgt 18 68 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 68 ctatctgacgttctctgt 18 69 19 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 69 tccatgacgt tcctgatgc 19 70 19DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 70 tccatgacgt tcctgatgc 19 71 19 DNA ArtificialSequence modified linkage of oligodeoxynucleotide phosphorothioate 71tccatgacgt tcctgatgc 19 72 19 DNA Artificial Sequence modified linkageof oligodeoxynucleotide phosphorothioate 72 tccatgacgg tcctgatgc 19 7316 DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 73 gagaacgctc gacctt 16 74 32 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 74 gagaacgctcgaccttgaga acgctcgacc tt 32 75 32 DNA Artificial Sequence modifiedlinkage of oligodeoxynucleotide phosphorothioate 75 ttccagctcgcaagaggaga acgctcgacc tt 32 76 32 DNA Artificial Sequence modifiedlinkage of oligodeoxynucleotide phosphorothioate 76 gagaacgctcgaccttttcc agctcgcaag ag 32 77 18 DNA Artificial Sequence modifiedlinkage of oligodeoxynucleotide phosphorothioate 77 tctcccagcg tgcgccat18 78 32 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 78 tcccagcgtg cgccattcccagcgtgcgcc at 32 79 32 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 79 taccgcgtgc gacccttcccagcgtgcgcc at 32 80 32 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 80 tcccagcgtg cgccattaccgcgtgcgacc ct 32 81 18 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 81 ctatctgacg ttctctgt 18 82 18DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 82 ctatctgacg ttctctgt 18 83 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 83 ctatctgacgttctctgt 18 84 18 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 84 ctatctgacg ttctctgt 18 85 18DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 85 ctatctgacg ttctctgt 18 86 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 86 ctatctgacgttctctgt 18 87 18 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 87 ctatctgacg ttctctgt 18 88 18DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 88 ctatctgacg ttctctgt 18 89 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 89 ctatctgacgttctctgt 18 90 18 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 90 ctatctgacg ttctctgt 18 91 18DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 91 cctactagcg ttctcatc 18 92 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 92 cctactagcgttctcatc 18 93 18 DNA Artificial Sequence modified linkage ofoligodeoxynucleotide phosphorothioate 93 cctactagcg ttctcatc 18 94 18DNA Artificial Sequence modified linkage of oligodeoxynucleotidephosphorothioate 94 cctactagcg ttctcatc 18 95 18 DNA Artificial Sequencemodified linkage of oligodeoxynucleotide phosphorothioate 95 cctactaggcttctcatc 18 96 18 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 96 ctatctgacg ttctctgt 18 97 18 DNA Artificial Sequencemodified oligodeoxynucleotide phosphorothioate 97 ctatctgagc ttctctgt 1898 18 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 98 tctcccagcg tgcgccat 18 99 18 DNA Artificial Sequencemodified oligodeoxynucleotide phosphorothioate 99 tctcccagcg tgcgccat 18100 18 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 100 ctatctgacg ttctctgt 18 101 18 DNA ArtificialSequence modified oligodeoxynucleotide phosphorothioate 101 ctatctgacgttctctgt 18 102 18 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 102 ctatctgagc ttctctgt 18 103 18 DNA ArtificialSequence modified oligodeoxynucleotide phosphorothioate 103 ctatctgacgttctctgt 18 104 18 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 104 ctatctgacg ttctctgt 18 105 18 DNA ArtificialSequence modified oligodeoxynucleotide phosphorothioate 105 ctatctgacgttctctgt 18 106 19 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 106 ctactctgac cttctctgt 19 107 18 DNA ArtificialSequence modified oligodeoxynucleotide phosphorothioate 107 ctatctgacgttctctgt 18 108 18 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 108 ctatctgacg ttctctgt 18 109 18 DNA ArtificialSequence modified oligodeoxynucleotide phosphorothioate 109 ctatctgacgttctctgt 18 110 18 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 110 ctatctgacg ttctctgt 18 111 18 DNA ArtificialSequence modified oligodeoxynucleotide phosphorothioate 111 ctatctgacgttctctgt 18 112 18 DNA Artificial Sequence modified oligodeoxynucleotidephosphorothioate 112 ctatctgacg ttctctgt 18

1-11. (Cancelled)
 12. An immunostimulatory oligonucleotide compoundcomprising a sequence of formula (III): (III) 5′-Um . . .U1-X1-X2-Y-Z-X3-X4-D1 . . . Dm-3′

wherein: Y is a non-natural pyrimidine nucleoside; Z is guanosine,2′-deoxy-guanosine or a non-natural purine nucleoside; each Xindependently is a naturally occurring nucleoside or animmunostimulatory moiety; wherein Um-U1 represents an upstreampotentiation domain, where each U independently is a naturally occurringnucleoside or an immunostimulatory moiety; wherein D1-Dm represents adownstream potentiation domain, where each D independently is anaturally occurring nucleoside or an immunostimulatory moiety; and m, ateach occurrence, represents a number from 0 to
 30. 13. Theimmunostimulatory oligonucleotide compound of claim 12, wherein at leastone X, U, or D is an immunostimulatory moiety.
 14. The immunostimulatoryoligonucleotide compound of claim 13, wherein: X1 is a naturallyoccurring nucleoside or an immunostimulatory moiety selected from thegroup consisting of C3-alkyl linker, 2-aminobutyl-1,3-propanediollinker, and P-L-deoxynucleoside; X2 is a naturally occurring nucleosideor an immunostimulatory moiety that is an amino linker; X3 is anaturally occurring nucleoside an immunostimulatory moiety that is anucleoside methylphosphonate; X4 is a naturally occurring nucleoside animmunostimulatory moiety selected from the group consisting ofnucleoside methylphosphonate and 2′-O-methyl-ribonucleoside; U1 is anaturally occurring nucleoside an immunostimulatory moiety selected fromthe group consisting of 1′,2′-dideoxyribose, C3-linker, and2′-O-methyl-ribonucleoside; U2 is a naturally occurring nucleoside animmunostimulatory moiety selected from the group consisting of1′,2′-dideoxyribose, C3-linker, Spacer 18, 3′-deoxy-nucleoside,nucleoside methylphosphonate, P-L-deoxynucleoside, and2′-O-propargylribonucleoside; U3 is a naturally occurring nucleoside animmunostimulatory moiety selected from the group consisting of1′,2′-dideoxyribose, C3-linker, Spacer 9, Spacer 18, nucleosidemethylphosphonate, and 2′-5′ linkage; D1 is a naturally occurringnucleoside an immunostimulatory moiety selected from the groupconsisting of 1′,2′-dideoxyribose and nucleoside methylphosphonate; D2is a naturally occurring nucleoside an immunostimulatory moiety selectedfrom the group consisting of 1′,2′-dideoxyribose, C3-linker, Spacer 9,Spacer 18, 2-aminobutyl-1,3-propanediol linker, nucleosidemethylphosphonate, and P-L-deoxynucleoside; and D3 is a naturallyoccurring nucleoside an immunostimulatory moiety selected from the groupconsisting of 3′-deoxynucleoside, 2′-O-propargylribonucleoside; and2′-5′ linkage.
 15. The immunostimulatory oligonucleotide compound ofclaim 13, wherein U2 and U3 are both the same immunostimulatory moietyselected from the group consisting of 1′,2′-didoxyribose, C3-linker, orβ-L-deoxynucleoside.
 16. The immunostimulatory oligonucleotide compoundof claim 13, wherein U3 and U4 are both the same immunostimulatorymoiety selected from the group consisting of nucleosidemethylphosphonate and 2′-O-methoxyethylribonucleoside.
 17. Theimmunostimulatory oligonucleotide compound of claim 13, wherein U5 andU6 are both the same immunostimulatory moiety selected from the groupconsisting of 1′,2′-dideoxyribose and C3-linker.
 18. Theimmunostimulatory oligonucleotide compound of claim 13, wherein X1 andU3 are both 1′,2′-dideoxyribose.
 19. The immunostimulatoryoligonucleotide compound of claim 13, wherein D2 and D3 are both thesame immunostimulatory moiety selected from the group consisting of1′,2′-dideoxyribose and β-L-deoxynucleoside. 20-38. (Cancelled)